Mitosis and meiosis are in many ways opposite processes. A principal role of DNA recombination in mitotic cells is to preserve the fidelity of genetic information and ensure that it is faithfully reproduced and passed on to daughter cells. In contrast, DNA recombination during meiosis acts to create new permutations of genetic information by facilitating reshuffling or intermixing of the maternal and paternal genomes during gamete formation to enable production of offspring with novel genomes as compared to either parent. The different purposes of DNA recombination in meiotic versus mitotic cells are reflected in the very different rolls and mechanisms of homologous recombination in each cell type [1-5; 7; 8].
There is a fundamental mechanistic distinction between the primary processes of homologous recombination in meiotic (germ-line) cells compared to mitotic (vegetative/somatic) cells. In meiotic cells, homologous recombination occurs primarily between non-sister chromatids (to shuffle the genome), whereas in mitotic cells homologous recombination occurs primarily between sister chromatids (to correct genomic errors). Sister chromatids are replicated copies of a particular maternal or paternal chromosome. Recombination between non-sister chromatids (i.e. between a paternal chromatid and a maternal chromatid) occurs 500-1000 fold more frequently in meiotic cells versus mitotic cells [48;50]. The meiotic process of non-sister chromatid exchange (NSCE) facilitates novel recombination of the genetic information from two parents of the organism. In contrast, the mitotic process of sister-chromatid exchange (SCE) resulting from recombination-mediated repair is a primary mechanism for maintaining genome fidelity throughout a multi-cellular organism.
There are a significant number of mechanical distinctions between mitotic SCE and meiotic NSCE, as these processes are currently understood. Physical interactions and recombination between meiotic chromosomes is associated with formation and function of the synaptonemal complex which is a unique proteinaceous structure that assembles during meiosis and participates in enabling pairing and exchange between non-sister chromatids [156; 157; 158; 159; 160]. Double-strand breaks in meiotic recombination are understood to be catalysed by a conserved, specific enzyme, SPO11 [4;9-11], whereas in mitotic cells double-strand breaks generally result from spontaneous lesions [3;7]. Upon formation of double strand breaks in either SCE or NSCE, the exposed double-stranded ends are understood to be resected by an exonuclease activity that degrades the DNA to generate single-stranded DNA (ssDNA) ends which have a 3′-hydroxyl group. This resection process is understood to be catalysed by a protein complex composed of at least three known proteins, MRE11, RAD50 and XRS2/NBS1 which are conserved from yeast to plants and humans [12-19]. The ssDNA ends may then be acted upon by another set of proteins so that the ends invade the sister chromatid in mitotic cells or, uniquely, the chromatid of the paired homologous chromosome from the other parent in meiotic cells.
Strand invasion may be catalysed by a group of proteins which are known as RecA homologues as a consequence of their sequence and functional similarity to the Escherichia coli RecA protein. RecA has been extensively studied and has been demonstrated in vitro to catalyse pairing between homologous DNA molecules and strand invasion [6]. Yeast are reported to have at least four proteins with homology to RecA: RAD51; RAD55; RAD57; and DMC1 [5]. These proteins are also highly conserved in plants and humans [21;22;24;25;39]. Eukaryotic RecA homologues also catalyse pairing between homologous DNA molecules and strand invasion [51;52]. Genetic studies illustrate the primacy of this group of proteins in mitotic and meiotic homologous recombination [13;20;23;53-57]. These biochemical and genetic studies demonstrate the high conservation of function of RecA homologues from lower to higher eukaryotes. Whereas RAD51, RAD55 and RAD57 play a role in both mitotic and meiotic homologous recombination [23;56], DMC1 functions in a meiosis-specific manner [20;54;55]. Biochemical and genetic evidence points to RAD51, RAD55 and RAD57 interacting in a common pathway whereas DMC1 acts in a unique but overlapping pathway [53;56]. The existence of two unique pathways of RecA homologues acting during meiosis is also supported by cytological studies whereby DMC1 and RAD51 are found at different nodes along the chromosome undergoing recombination [53]. RAD51, RAD55 and RAD57 may only facilitate homologous recombination on DNA molecules with a specific structure and topology unique to this group of proteins [59]. It has been proposed that DMC1 may act on specific DNA structures, potentially not recognized by RAD51, RAD55 or RAD57, to promote pairing and recombination between homologous maternal and paternal chromosomes and catalyse NSCE [53;60]. These DNA structures may be meiosis-specific, again illustrating the unique attributes of homologous recombination involving NSCE during meiosis versus SCE in mitotic cells.
While RAD51 and DMC1 can apparently catalyse pairing and strand invasion alone, they also act in concert with other proteins that enhance homologous recombination. For example, RAD51 physically interacts with RAD54 [61;62] and RAD52 [42] and both of these proteins are conserved from yeast to humans [64;65]. Inclusion of RAD54 or RAD52 in in vitro assays demonstrate these proteins can stimulate the pairing and strand invasion activity of RAD51 [23;66]. DMC1 does not physically interact with RAD54 [53] but does interact with a RAD54 homologue, known as TID1 [53], which acts in NSCE during meiosis [49]. This again illustrates the uniqueness of the homologous recombination pathways catalysed by DMC1 versus RAD51. In addition to the promoting effects of RAD54 and RAD52, homologous recombination is enhanced by a complex of proteins which bind ssDNA. In eukaryotes, this protein complex is a heterotrimer known as RPA [26]. ssDNA-binding proteins function in DNA recombination and repair by reducing secondary structure in ssDNA thereby increasing the ability of RecA-like proteins to bind and act upon the ssDNA [67]. RPA is conserved from yeast to humans [26]. RPA has been demonstrated to physically interact with RAD51 and DMC1 [68], as well as associating with RAD52 [69], and may thereby act in recruiting RecA-homologues and/or other recombination proteins to recombinogenic ends, or assist in forming recombinogenic DNA-protein complexes.
Other participants in the pairing and strand exchange processes leading to homologous recombination in meiotic cells include MSH4, MSH5 and MLH1[27;29;31]. These proteins are also conserved from yeast to plants and humans [28;30;32;33]. MLH1 functions principally in mismatch repair to ensure fidelity of DNA replication in vegetative cells but also plays a role in homologous recombination in meiotic cells [31]. MSH4 and MSH5 are meiosis-specific homologues of a set of proteins, unique from MLH1, which function in mismatch repair in vegetative cells [27; 29]. The biochemical role of MSH4 and MSH5 during meiosis is unclear as yet but evidence points to these proteins participating in DNA exchange between homologous chromosomes [27; 29]. The specificity of MSH4 and MSH5 to homologous recombination in meiotic cells again points to the uniqueness of this homologous recombination process versus that which occurs in vegetative cells.
Strand invasion and formation of the initial crossover or chiasma between the sister chromatids in vegetative cells and non-sister chromatids in meiotic cells is followed by branch migration, DNA replication and strand displacement. This increases the length of genetic information exchanged between the two chromatids. A second chiasma then occurs. The chiasma are acted upon by an enzyme known as a resolvase. This family of enzymes recognize and bind the cruciform structure created by the chiasma between the paired chromatids. Resolvases have been well characterized in microorganisms, including lower eukaryotes[43; 44], and the activity has been detected in humans [161].
It has been suggested that recombinases may be used to stimulate mitotic homologous recombination between sister chromatids in eukaryotes, which has been proposed as a mechanism to promote gene targeting in vegetative/somatic cells [63;82;85;86]. Gene targeting generally involves the directed alteration of a specific DNA sequence in its genomic locus in vivo. Problems have however been reported with mitotic gene targeting. It has for example been found that overexpression of RecA-homologues in mitotic cells may cause cell cycle arrest [92]. International Patent Publication WO 97/08331 dated 6 Mar. 1997 summarizes a range of difficulties with earlier suggestions that the E. coli RecA recombinase would be useful for stimulating homologous mitotic recombination (as for example had been suggested in International Patent Publications WO 93/22443, WO 94/04032 and WO 93/06221). Utilization of E. coli RecA in eukaryotic cells is potentially problematic because the direction of strand transfer catalysed by RecA is the opposite to the direction of strand transfer catalysed by eukaryotic RecA homologues [52]. Nevertheless, overexpression of E. coli RecA has been reported to promote gene targeting approximately 10-fold in mouse cells [63] and less than two-fold in plants [82]. However, this latter result in plants also demonstrated a very low overall frequency of gene targeting, which would tend to cast doubt on the statistical significance of the result.
In the face of difficulties associated with the use of E. coli RecA in mitotic gene targeting, alternative enzymes have been used to catalyse homologous sister chromatid exchange in mitotic cells. For example, U.S. Pat. Nos. 5,780,296 and 5,945,339 disclose methods to promote homologous recombination using Rec2 as an alternative recombinase to overcome problems with the use of RecA [86]. It has been reported that overexpression of human RAD51 (hRAD51) can increase gene targeting frequency by 2-3 fold [85].
In applications other than gene targeting in mitotic cells, other studies have suggested that increased expression of E. coli RecA or RAD51 may increase the resistance of cells to radiation or other DNA damaging agents [82; 85; 87-89; 91], and enhance the frequency of intrachromosomal recombination [88;90;91] and sister-chromatid exchange [82]. It has also been suggested that increased RAD51 activity during meiosis has no effect on the viability of gametes, although no evaluation of homologous recombination in these cells was conducted [87]. Identification of mechanistic steps in meiotic homologous recombination has utilized genetic analysis of mutants to identify genes involved in homologous recombination and DNA repair, and mutants with reduced meiotic homologous recombination have been identified [9;20;23;93]. Null-mutations typically have a severe effect on the whole meiotic process, and can affect viability of gametes [9;20;54;55;93-95] and have pleiotropic effects on the organism at different developmental stages or in different tissues or in response to environmental conditions. For example, rad51 null mutants may have decreased meiotic homologous recombination frequency but they also reportedly have poor DNA repair and resistance to environmental stresses and DNA damaging agents [96;97], as well as a lethal phenotype in embryos [95].